Engineered enzyme having acetoacetyl-coa hydrolase activity, microorganisms comprising same, and methods of using same

ABSTRACT

The disclosure provides engineered enzymes that are capable of mediating the conversion of acetoacetyl-CoA to acetoacetate that do not react with the same order of magnitude with acetyl-CoA as they do with acetoacetyl-CoA (e.g., the engineered enzymes have a specific acetoacetyl-CoA hydrolase activity at least 10x higher than its acetyl-CoA hydrolase activity). Additionally, the disclosure provides modified microorganisms that comprise the engineered enzymes disclosed herein and methods of using same.

BACKGROUND

The conversion of acetoacetyl-CoA to acetoacetate (FIG. 1) is anessential step in metabolic pathways with such intermediates. Thespecific hydrolysis of the thioester bond between coenzyme A (a thiol)and acetoacetate (an acyl group carrier) in acetoacetyl-CoA is anefficient way to produce the aforementioned conversion. Two classes ofnaturally occurring enzymes have been used to mediate such conversionincluding, CoA transferases (E.C. 2.8.3.-) and CoA-hydrolases(thioesterases) (E.C. 3.1.2.-). However, while acetoacetate-CoAtransferases require the presence of a non-activated acid acting as CoAacceptor, the CoA-hydrolases (acyl-CoA thioesterases) described to acton acetoacetyl-CoA are unspecific in the sense that they react with thesame order of magnitude with acetyl-CoA, the substrate required foracetoacetyl-CoA formation by the enzyme thiolase (E.C.2.3.1.9), therebydegrading the substrate for the acetoacetyl-CoA biosynthesis itself.

Therefore, there exists a need in the art for improved enzymes tomediate the conversion of acetoacetyl-CoA to acetoacetate.

SUMMARY

The present disclosure provides engineered enzymes that are capable ofmediating the conversion of acetoacetyl-CoA to acetoacetate that do notreact with the same order of magnitude with acetyl-CoA as they do withacetoacetyl-CoA (e.g., the engineered enzymes have a specificacetoacetyl-CoA hydrolase activity at least 10× higher than itsacetyl-CoA hydrolase activity).

The present disclosure also provides an engineered enzyme havingacetoacetyl-CoA substrate specificity and acetoacetyl-CoA specifichydrolase activity.

In some embodiments of each or any of the above or below mentionedembodiments, the engineered enzyme comprises i) an amino acid sequenceof an enzyme having acetoacetyl-CoA transferase activity; and ii) asubstitution of a glutamic acid residue (i.e., the catalytic glutamicacid residue) to an aspartic acid residue at a position corresponding toamino acid position 51 of SEQ ID NO: 1, or a substitution of a glutamicacid residue to an aspartic acid residue at a position corresponding toamino acid position 46 of SEQ ID NO: 3, or a substitution of a glutamicacid residue to an aspartic acid residue at a position corresponding toamino acid position 333 of SEQ ID NO: 5.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme having acetoacetyl-CoA transferase activity isfrom an enzyme family having 3-oxoacid CoA-transferase activity.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme having acetoacetyl-CoA transferase activity isbutyrate-acetoacetate CoA-transferase or acetate-acetoacetate-CoAtransferase.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme having acetoacetyl-CoA transferase activity isfrom Clostridium acetobutilicum.

In some embodiments of each or any of the above or below mentionedembodiments, the engineered enzyme has the amino acid sequence as setforth in SEQ ID NO: 2.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme having acetoacetyl-CoA transferase activity isfrom Escherichia coli.

In some embodiments of each or any of the above or below mentionedembodiments, the engineered enzyme has the amino acid sequence as setforth in SEQ ID NO: 3.

In some embodiments of each or any of the above or below mentionedembodiments, the engineered enzyme has the amino acid sequence as setforth in SEQ ID NO: 5.

In some embodiments of each or any of the above or below mentionedembodiments, the engineered enzyme has a specific acetoacetyl-CoAhydrolase activity at least 10× higher than its acetyl-CoA hydrolaseactivity.

The present disclosure also provides an engineered enzyme having theamino acid sequence as set forth in SEQ ID NO: 2.

The present disclosure also provides a modified microorganism comprisingone or more polynucleotides coding for one or more enzymes in a pathwaywith acetoacetate as an intermediate or end-product, and an engineeredenzyme having acetoacetyl-CoA substrate specificity and acetoacetyl-CoAspecific hydrolase activity.

In some embodiments of each or any of the above or below mentionedembodiments, the microorganism has a disruption in one or morepolynucleotides that code for one or more enzymes that decarboxylatepyruvate or a disruption in one or more polynucleotides that code for atranscription factor of an enzyme that decarboxylates pyruvate.

In some embodiments of each or any of the above or below mentionedembodiments, the disruption in the one or more enzymes thatdecarboxylate pyruvate is a deletion or a mutation.

In some embodiments of each or any of the above or below mentionedembodiments, the one or more enzymes that decarboxylate pyruvate includepdc1, pdc 5, and/or pdc6, and wherein the one or more transcriptionfactors of the one or more enzymes that decarboxylate pyruvate includepdc2.

In some embodiments of each or any of the above or below mentionedembodiments, the engineered enzyme comprises i) an amino acid sequenceof an enzyme having acetoacetyl-CoA transferase activity and ii) asubstitution of a glutamic acid residue to an aspartic acid residue at aposition corresponding to amino acid position 51 of SEQ ID NO: 1.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme having acetoacetyl-CoA transferase activity isfrom an enzyme family having 3-oxoacid CoA-transferase activity.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme having acetoacetyl-CoA transferase activity isbutyrate-acetoacetate CoA-transferase or acetate-acetoacetate-CoAtransferase.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme having acetoacetyl-CoA transferase activity isfrom Clostridium acetobutilicum.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme has the amino acid sequence as set forth in SEQID NO: 2.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme having acetoacetyl-CoA transferase activity isfrom Escherichia coli.

In some embodiments of each or any of the above or below mentionedembodiments, the engineered enzyme has the amino acid sequence as setforth in SEQ ID NO: 4.

In some embodiments of each or any of the above or below mentionedembodiments, the engineered enzyme has the amino acid sequence as setforth in SEQ ID NO: 6.

In some embodiments of each or any of the above or below mentionedembodiments, the engineered enzyme has a specific acetoacetyl-CoAhydrolase activity at least 10× higher than its acetyl-CoA hydrolaseactivity.

The present disclosure also provides a method of engineering an enzymehaving acetoacetyl-CoA substrate specificity and acetoacetyl-CoAspecific hydrolase activity, the method comprising: a) selecting anenzyme having acetoacetyl-CoA transferase activity, and b) substitutinga glutamic acid residue to an aspartic acid residue at a positioncorresponding to amino acid position 51 of SEQ ID NO: 1 in the enzymehaving acetoacetyl-CoA transferase activity to produce an engineeredenzyme.

In some embodiments of each or any of the above or below mentionedembodiments, the substitution is introduced via site directedmutagenesis.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme having acetoacetyl-CoA transferase activity isfrom an enzyme family having 3-oxoacid CoA-transferase activity.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme having acetoacetyl-CoA transferase activity isbutyrate-acetoacetate CoA-transferase.

In some embodiments of each or any of the above or below mentionedembodiments, the enzyme having acetoacetyl-CoA transferase activity isfrom Clostridium acetobutilicum or Escherichia coli.

In some embodiments of each or any of the above or below mentionedembodiments, the engineered enzyme has a specific acetoacetyl-CoAhydrolase activity at least 10× higher than its acetyl-CoA hydrolaseactivity.

The present disclosure also provides a method of producing one or moreproducts from a fermentable carbon source, said method comprising: a.)providing a fermentable carbon source; and b.) contacting thefermentable carbon source with the modified microorganism as disclosedherein in a fermentation media, wherein the microorganism produces oneor more products from the fermentable carbon source.

In some embodiments of each or any of the above or below mentionedembodiments, the carbon source is contacted with the modifiedmicroorganism under anaerobic conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe disclosure, will be better understood when read in conjunction withthe appended figures. For the purpose of illustrating the disclosure,shown in the figures are embodiments which are presently preferred. Itshould be understood, however, that the disclosure is not limited to theprecise arrangements, examples and instrumentalities shown.

FIG. 1 depicts a reaction scheme for the formation of acetoacetatethrough hydrolysis of acetoacetyl-CoA.

FIG. 2 depicts reaction schemes for metabolic pathways with theintermediates acetoacetyl-CoA and acetoacetate.

FIG. 3 depicts an alignment of 3-oxoacid CoA-transferases illustratingthe identification and location of the active glutamic acid residue.

FIG. 4 depicts an exemplary pathway for the co-production of 1-propanoland 2-propanol, where 1-propanol is produced via adihydroxyacetone-phosphate intermediate.

FIG. 5 depicts an exemplary pathway for the co-production of 1-propanoland 2-propanol, where 1-propanol is produced via a glyceraldehyde3-phosphate.

DETAILED DESCRIPTION

The conversion of acetoacetyl-CoA to acetoacetate (FIG. 1) is anessential step in metabolic pathways with such intermediates including,for example, pathways for the production of 3-hydroxy-butyrate, acetoneor isopropanol (FIG. 2). However, no acetoacetyl-CoA specific hydrolaseis known that can produce acetoacetate and regenerate free CoA withoutdegrading acetyl-CoA, the substrate for the acetoacetyl-CoA biosynthesisitself. The present disclosure provides the rational engineering of a3-oxoacid CoA-transferase with acetoacetyl-CoA substrate specificity(e.g., a butyrate-acetoacetate CoA-transferase—SEQ ID NO: 1; aacetate-acetoacetate CoA-transferase—SEQ ID NO: 2; or AcetateCoA-transferase—SEQ ID NO: 3) to an acetoacetyl-CoA specific hydrolaseand its use in metabolic pathways utilizing acetoacetate as anintermediate or an end-product including, for example, pathways for thesynthesis of 3-hydroxy-butyrate, acetone and/or isopropanol. Theengineered enzyme has a higher activity on acetoacetyl-CoA versusacetyl-CoA (e.g., 10×, 15×, 20×, 25×, 30×, 35×, 40×, 45×, 50×, or more).The engineered enzyme may comprise: i) an amino acid sequence of anenzyme having acetoacetyl-CoA transferase activity and ii) asubstitution of a glutamic acid residue to an aspartic acid residue at aposition corresponding to amino acid position 51 of SEQ ID NO: 1, or asubstitution of a glutamic acid residue to an aspartic acid residue at aposition corresponding to amino acid position 46 of SEQ ID NO: 3, or asubstitution of a glutamic acid residue to an aspartic acid residue at aposition corresponding to amino acid position 333 of SEQ ID NO: 5; andhave a specific acetoacetyl-CoA hydrolase activity at least 10× higherthan its acetyl-CoA hydrolase activity. Exemplary 3-oxoacidCoA-transferases are listed in FIG. 3 as well as the location of theactive glutamic acid residue that may be substituted to an aspartic acidresidue to engineer an enzyme having acetoacetyl-CoA specific hydrolaseactivity.

The engineered acetoacetyl-CoA hydrolase disclosed herein solves theproblem that no acetoacetyl-CoA specific hydrolase is known that canproduce acetoacetate and regenerate free CoA. Natural wild typehydrolases are known to accept several acid CoA compounds with similaractivities and can be expected to be very difficult to be engineered forsuch specificity. Thus, naturally occurring and known enzymes withacetoacetyl-CoA hydrolase activity suggested previously (see, US2010/0261237 A1) create the problem of unspecific acid-CoA (e.g.,acetyl-CoA) hydrolase activity. Such enzymes destroy the precursornecessary for the formation of their own substrate, i.e. acetoacetyl-CoAgeneration from two acetyl-CoA by thiolase enzyme. As a result, theiruse in metabolic pathways containing further acid-CoA intermediates ishighly inefficient.

Additionally, the engineered acetoacetyl-CoA hydrolase disclosed hereinsolves the problem of requiring an acceptor molecule and processinganother acid-CoA intermediate. Appropriate transferase enzymes for theenzymatic removal of the CoA group from acetoacetyl-CoA are typicallyspecific. However, the reaction requires an acceptor acid molecule andyields a further acid-CoA compound that needs to be processed forregeneration of free CoA. Removing the necessity of an acceptor moleculeenables the creation of simplified, usually more efficient pathways.

Furthermore, transferases already described to accept acetoacetyl-CoA assubstrate have a Km value for the acceptor molecule that is above 10 mMwhich is about 1000 times higher than the Km value for theacetoacetyl-CoA donor substrate. So the acceptor concentration is alimiting factor of the transferase reaction. Utilizing anacetoacetyl-CoA hydrolase engineered from an acetoacetyl-CoA transferase(i.e., a 3-oxoacid CoA-transferase that accepts Acetoacetyl-CoA as a CoAdonor) has the added benefit of a very low Km for the substrateacetoacetyl-CoA. This allows hydrolysis of acetoacetyl-CoA with a highreaction rate at low substrate concentrations and therefore can preventaccumulation of acetoacetyl-CoA and establish a “pull” on the preceding,thiolase mediated reversible acetoacetyl-CoA biosynthesis reaction.Since the thiolase reaction often represents a rate limiting step in abiosynthesis, such a pull can be highly beneficial for the performanceof the entire appropriate metabolic pathway.

The invention disclosed herein has particular importance in the contextof a microorganism such as Saccharomyces cerevisiase strain that has thepyruvate decarbolylase genes (e.g., PDC1, PDC5 and PDC6) disruptedand/or deleted. In this strain, the reaction catalyzed by this enzyme,namely the conversion of pyruvate to acetaldehyde and CO₂ does notoccur. The result of such a deletion is that acetaldehyde cannot befurther reduced by alcohol dehydrogenase to make ethanol, and thus suchstrain is deemed ethanol null. A secondary effect of such a deletion isthat such a strain also does not produce acetic acid, which in the2-propanol plathway described herein (see, e.g., Table 3 and FIGS. 4 and5), is an essential receptor for a CoA which is transferred fromacetoacetyl-CoA as it is converted to acetoacetate by a transferase.Thus, in the absence of a CoA receptor for such a reaction, it isimpossible to remove the CoA from acetoacetyl-CoA, and the pathwaycannot advance to 2-propanol. Pyruvate decarbolylase null yeast strainsmodified to produce 2-propanol thus require either exogenous acetate toreceive the CoA from acetoacetyl-CoA, or require the activity of anenzyme such as a hydrolase to remove such a CoA from acetoacetyl-CoA.The hydrolase thus proposed has practical application in the context ofsuch a strain which is unable to produce acetic acid, but requires amanner to convert acetoacetyl-CoA to acetoacetate.

Microorganisms disclosed herein with an engineered acetoacetyl-CoAspecific hydrolase may also be modified to have a disruption in one ormore polynucleotides that code for one or more enzymes thatdecarboxylate pyruvate or a disruption in one or more polynucleotidesthat code for a transcription factor of an enzyme that decarboxylatespyruvate. In an embodiment, the disruption in the one or more enzymesthat decarboxylate pyruvate is a deletion or a mutation. In a furtherembodiment, the one or more enzymes that decarboxylate pyruvate includepdc1, pdc 5, and/or pdc6, and the one or more transcription factors ofthe one or more enzymes that decarboxylate pyruvate include pdc2. Themicroorganism may additionally comprise one or more exogenouspolynucleotides encoding one or more enzymes in pathways for theco-production of 1-propanol and/or 2-propanol from a fermentable carbonsource under anaerobic conditions.

As used herein, the term “biological activity” or “functional activity,”when referring to a protein, polypeptide or peptide, may mean that theprotein, polypeptide or peptide exhibits a functionality or propertythat is useful as relating to some biological process, pathway orreaction. Biological or functional activity can refer to, for example,an ability to interact or associate with (e.g., bind to) anotherpolypeptide or molecule, or it can refer to an ability to catalyze orregulate the interaction of other proteins or molecules (e.g., enzymaticreactions).

As used herein, the term “culturing” may refer to growing a populationof cells, e.g., microbial cells, under suitable conditions for growth,in a liquid or on solid medium.

As used herein, the term “derived from” may encompass the termsoriginated from, obtained from, obtainable from, isolated from, andcreated from, and generally indicates that one specified material findsits origin in another specified material or has features that can bedescribed with reference to the another specified material.

As used herein, “exogenous polynucleotide” refers to anydeoxyribonucleic acid that originates outside of the microorganism.

As used herein, the term “an expression vector” may refer to a DNAconstruct containing a polynucleotide or nucleic acid sequence encodinga polypeptide or protein, such as a DNA coding sequence (e.g. genesequence) that is operably linked to one or more suitable controlsequence(s) capable of affecting expression of the coding sequence in ahost. Such control sequences include a promoter to affect transcription,an optional operator sequence to control such transcription, a sequenceencoding suitable mRNA ribosome binding sites, and sequences whichcontrol termination of transcription and translation. The vector may bea plasmid, cosmid, phage particle, bacterial artificial chromosome, orsimply a potential genomic insert. Once transformed into a suitablehost, the vector may replicate and function independently of the hostgenome (e.g., independent vector or plasmid), or may, in some instances,integrate into the genome itself (e.g., integrated vector). The plasmidis the most commonly used form of expression vector. However, thedisclosure is intended to include such other forms of expression vectorsthat serve equivalent functions and which are, or become, known in theart.

As used herein, the term “expression” may refer to the process by whicha polypeptide is produced based on a nucleic acid sequence encoding thepolypeptides (e.g., a gene). The process includes both transcription andtranslation.

As used herein, the term “gene” may refer to a DNA segment that isinvolved in producing a polypeptide or protein (e.g., fusion protein)and includes regions preceding and following the coding regions as wellas intervening sequences (introns) between individual coding segments(exons).

As used herein, the term “heterologous,” with reference to a nucleicacid, polynucleotide, protein or peptide, may refer to a nucleic acid,polynucleotide, protein or peptide that does not naturally occur in aspecified cell, e.g., a host cell. It is intended that the termencompass proteins that are encoded by naturally occurring genes,mutated genes, and/or synthetic genes. In contrast, the term homologous,with reference to a nucleic acid, polynucleotide, protein or peptide,refers to a nucleic acid, polynucleotide, protein or peptide that occursnaturally in the cell.

As used herein, the term a “host cell” may refer to a cell or cell line,including a cell such as a microorganism which a recombinant expressionvector may be transfected for expression of a polypeptide or protein(e.g., fusion protein). Host cells include progeny of a single hostcell, and the progeny may not necessarily be completely identical (inmorphology or in total genomic DNA complement) to the original parentcell due to natural, accidental, or deliberate mutation. A host cell mayinclude cells transfected or transformed in vivo with an expressionvector.

As used herein, the term “introduced,” in the context of inserting anucleic acid sequence or a polynucleotide sequence into a cell, mayinclude transfection, transformation, or transduction and refers to theincorporation of a nucleic acid sequence or polynucleotide sequence intoa eukaryotic or prokaryotic cell wherein the nucleic acid sequence orpolynucleotide sequence may be incorporated into the genome of the cell(e.g., chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed.

As used herein, the term “non-naturally occurring” or “modified” whenused in reference to a microbial organism or microorganism of theinvention is intended to mean that the microbial organism has at leastone genetic alteration not normally found in a naturally occurringstrain of the referenced species, including wild-type strains of thereferenced species. Genetic alterations include, for example,modifications introducing expressible nucleic acids encoding metabolicpolypeptides, other nucleic acid additions, nucleic acid deletionsand/or other functional disruption of the microbial organism's geneticmaterial. Such modifications include, for example, coding regions andfunctional fragments thereof, for heterologous, homologous or bothheterologous and homologous polypeptides for the referenced species.Additional modifications include, for example, non-coding regulatoryregions in which the modifications alter expression of a gene or operon.Non-naturally occurring microbial organisms of the disclosure cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely. Those skilled in the art willunderstand that the genetic alterations, including metabolicmodifications exemplified herein, are described with reference to asuitable host organism such as E. coli and their corresponding metabolicreactions or a suitable source organism for desired genetic materialsuch as genes for a desired metabolic pathway. However, given thecomplete genome sequencing of a wide variety of organisms and the highlevel of skill in the area of genomics, those skilled in the art willreadily be able to apply the teachings and guidance provided herein toessentially all other organisms. For example, the E. coli metabolicalterations exemplified herein can readily be applied to other speciesby incorporating the same or analogous encoding nucleic acid fromspecies other than the referenced species. Such genetic alterationsinclude, for example, genetic alterations of species homologs, ingeneral, and in particular, orthologs, paralogs or nonorthologous genedisplacements.

As used herein, the term “operably linked” may refer to a juxtapositionor arrangement of specified elements that allows them to perform inconcert to bring about an effect. For example, a promoter may beoperably linked to a coding sequence if it controls the transcription ofthe coding sequence.

As used herein, “1-propanol” is intended to mean n-propanol with ageneral formula CH₃CH₂CH₂OH (CAS number—71-23-8).

As used herein, “2-propanol” is intended to mean isopropyl alcohol witha general formula CH₃CH₃CHOH (CAS number—67-63-0).

As used herein, the term “a promoter” may refer to a regulatory sequencethat is involved in binding RNA polymerase to initiate transcription ofa gene. A promoter may be an inducible promoter or a constitutivepromoter. An inducible promoter is a promoter that is active underenvironmental or developmental regulatory conditions.

As used herein, the term “a polynucleotide” or “nucleic acid sequence”may refer to a polymeric form of nucleotides of any length and anythree-dimensional structure and single- or multi-stranded (e.g.,single-stranded, double-stranded, triple-helical, etc.), which containdeoxyribonucleotides, ribonucleotides, and/or analogs or modified formsof deoxyribonucleotides or ribonucleotides, including modifiednucleotides or bases or their analogs. Such polynucleiotides or nucleicacid sequences may encode amino acids (e.g., polypeptides or proteinssuch as fusion proteins). Because the genetic code is degenerate, morethan one codon may be used to encode a particular amino acid, and thepresent disclosure encompasses polynucleotides which encode a particularamino acid sequence. Any type of modified nucleotide or nucleotideanalog may be used, so long as the polynucleotide retains the desiredfunctionality under conditions of use, including modifications thatincrease nuclease resistance (e.g., deoxy, 2′-O-Me, phosphorothioates,etc.). Labels may also be incorporated for purposes of detection orcapture, for example, radioactive or nonradioactive labels or anchors,e.g., biotin. The term polynucleotide also includes peptide nucleicacids (PNA). Polynucleotides may be naturally occurring or non-naturallyoccurring. The terms polynucleotide, nucleic acid, and oligonucleotideare used herein interchangeably. Polynucleotides may contain RNA, DNA,or both, and/or modified forms and/or analogs thereof. A sequence ofnucleotides may be interrupted by non-nucleotide components. One or morephosphodiester linkages may be replaced by alternative linking groups.These alternative linking groups include, but are not limited to,embodiments wherein phosphate is replaced by P(O)S (thioate), P(S)S(dithioate), (O)NR₂ (amidate), P(O)R, P(O)OR′, COCH₂ (formacetal), inwhich each R or R′ is independently H or substituted or unsubstitutedalkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl,alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. Polynucleotides may be linear orcircular or comprise a combination of linear and circular portions.

As used herein, the term a “protein” or “polypeptide” may refer to acomposition comprised of amino acids and recognized as a protein bythose of skill in the art. The conventional one-letter or three-lettercode for amino acid residues is used herein. The terms protein andpolypeptide are used interchangeably herein to refer to polymers ofamino acids of any length, including those comprising linked (e.g.,fused) peptides/polypeptides (e.g., fusion proteins). The polymer may belinear or branched, it may comprise modified amino acids, and it may beinterrupted by non-amino acids. The terms also encompass an amino acidpolymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art.

As used herein, related proteins, polypeptides or peptides may encompassvariant proteins, polypeptides or peptides. Variant proteins,polypeptides or peptides differ from a parent protein, polypeptide orpeptide and/or from one another by a small number of amino acidresidues. In some embodiments, the number of different amino acidresidues is any of about 1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, or50. In some embodiments, variants differ by about 1 to about 10 aminoacids. Alternatively or additionally, variants may have a specifieddegree of sequence identity with a reference protein or nucleic acid,e.g., as determined using a sequence alignment tool, such as BLAST,ALIGN, and CLUSTAL (see, infra). For example, variant proteins ornucleic acid may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or even 99.5% amino acid sequence identity with areference sequence.

As used herein, the term “recovered,” “isolated,” “purified,” and“separated” may refer to a material (e.g., a protein, peptide, nucleicacid, polynucleotide or cell) that is removed from at least onecomponent with which it is naturally associated. For example, theseterms may refer to a material which is substantially or essentially freefrom components which normally accompany it as found in its nativestate, such as, for example, an intact biological system.

As used herein, the term “recombinant” may refer to nucleic acidsequences or polynucleotides, polypeptides or proteins, and cells basedthereon, that have been manipulated by man such that they are not thesame as nucleic acids, polypeptides, and cells as found in nature.Recombinant may also refer to genetic material (e.g., nucleic acidsequences or polynucleotides, the polypeptides or proteins they encode,and vectors and cells comprising such nucleic acid sequences orpolynucleotides) that has been modified to alter its sequence orexpression characteristics, such as by mutating the coding sequence toproduce an altered polypeptide, fusing the coding sequence to that ofanother coding sequence or gene, placing a gene under the control of adifferent promoter, expressing a gene in a heterologous organism,expressing a gene at decreased or elevated levels, expressing a geneconditionally or constitutively in manners different from its naturalexpression profile, and the like.

As used herein, the term “selective marker” or “selectable marker” mayrefer to a gene capable of expression in a host cell that allows forease of selection of those hosts containing an introduced nucleic acidsequence, polynucleotide or vector. Examples of selectable markersinclude but are not limited to antimicrobial substances (e.g.,hygromycin, bleomycin, or chloramphenicol) and/or genes that confer ametabolic advantage, such as a nutritional advantage, on the host cell.

As used herein, the term “substantially anaerobic” means that growth ofthe modified micororganism takes place in culture media that comprises adissolved oxygen concentration of less than 5 ppm.

As used herein, the term “substantially similar” and “substantiallyidentical” in the context of at least two nucleic acids,polynucleotides, proteins or polypeptides may mean that a nucleic acid,polynucleotide, protein or polypeptide comprises a sequence that has atleast about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even99.5% sequence identity, in comparison with a reference (e.g.,wild-type) nucleic acid, polynucleotide, protein or polypeptide.Sequence identity may be determined using known programs such as BLAST,ALIGN, and CLUSTAL using standard parameters. (See, e.g., Altshul et al.(1990) J. Mol. Biol. 215:403-410; Henikoff et al. (1989) Proc. Natl.Acad. Sci. 89:10915; Karin et al. (1993) Proc. Natl. Acad. Sci. 90:5873;and Higgins et al. (1988) Gene 73:237). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. Also, databases may be searched using FASTA(Person et al. (1988) Proc. Natl. Acad. Sci. 85:2444-2448.) In someembodiments, substantially identical polypeptides differ only by one ormore conservative amino acid substitutions. In some embodiments,substantially identical polypeptides are immunologically cross-reactive.In some embodiments, substantially identical nucleic acid moleculeshybridize to each other under stringent conditions (e.g., within a rangeof medium to high stringency).

As used herein, the term “transfection” or “transformation” may refer tothe insertion of an exogenous nucleic acid or polynucleotide into a hostcell. The exogenous nucleic acid or polynucleotide may be maintained asa non-integrated vector, for example, a plasmid, or alternatively, maybe integrated into the host cell genome. The term transfecting ortransfection is intended to encompass all conventional techniques forintroducing nucleic acid or polynucleotide into host cells. Examples oftransfection techniques include, but are not limited to, calciumphosphate precipitation, DEAE-dextran-mediated transfection,lipofection, electroporation, and microinjection.

As used herein, the term “transformed,” “stably transformed,” and“transgenic” may refer to a cell that has a non-native (e.g.,heterologous) nucleic acid sequence or polynucleotide sequenceintegrated into its genome or as an episomal plasmid that is maintainedthrough multiple generations.

As used herein, the term “vector” may refer to a polynucleotide sequencedesigned to introduce nucleic acids into one or more cell types. Vectorsinclude cloning vectors, expression vectors, shuttle vectors, plasmids,phage particles, single and double stranded cassettes and the like.

As used herein, the term “wild-type,” “native,” or “naturally-occurring”proteins may refer to those proteins found in nature. The termswild-type sequence refers to an amino acid or nucleic acid sequence thatis found in nature or naturally occurring. In some embodiments, awild-type sequence is the starting point of a protein engineeringproject, for example, production of variant proteins.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Singleton, et al.,Dictionary of Microbiology and Molecular Biology, second ed., John Wileyand Sons, New York (1994), and Hale & Markham, The Harper CollinsDictionary of Biology, Harper Perennial, N.Y. (1991) provide one ofskill with a general dictionary of many of the terms used in thisdisclosure. Further, it will be understood that any of the substratesdisclosed in any of the pathways herein may alternatively include theanion or the cation of the substrate.

Numeric ranges provided herein are inclusive of the numbers defining therange.

Unless otherwise indicated, nucleic acids sequences are written left toright in 5′ to 3′ orientation; amino acid sequences are written left toright in amino to carboxy orientation, respectively.

While the present disclosure is capable of being embodied in variousforms, the description below of several embodiments is made with theunderstanding that the present disclosure is to be considered as anexemplification of the disclosure, and is not intended to limit thedisclosure to the specific embodiments illustrated. Headings areprovided for convenience only and are not to be construed to limit thedisclosure in any manner. Embodiments illustrated under any heading maybe combined with embodiments illustrated under any other heading.

The use of numerical values in the various quantitative values specifiedin this application, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges were both preceded by the word “about.” Also, thedisclosure of ranges is intended as a continuous range including everyvalue between the minimum and maximum values recited as well as anyranges that can be formed by such values. Also disclosed herein are anyand all ratios (and ranges of any such ratios) that can be formed bydividing a disclosed numeric value into any other disclosed numericvalue. Accordingly, the skilled person will appreciate that many suchratios, ranges, and ranges of ratios can be unambiguously derived fromthe numerical values presented herein and in all instances such ratios,ranges, and ranges of ratios represent various embodiments of thepresent disclosure.

Engineering of Acetoacetyl-CoA Hydrolase

A transferase with acetoacetyl-CoA substrate specificity may beengineered to produce an acetoacetyl-CoA specific hydrolase. Thedisclosure contemplates that any method known in the art may be used tomodify a transferase with acetoacetyl-CoA substrate specificityincluding, for example, site directed mutagenesis. In an embodiment, thetransferase with acetoacetyl-CoA substrate specificity may be modifiedto comprise a substitution of a glutamic acid residue to an asparticacid residue at a position corresponding to amino acid position 51 ofSEQ ID NO: 1. The engineered enzyme may be subjected to furthermutagenesis (e.g., random mutagenesis) to further increase its hydrolaseactivity.

The present disclosure also provides a method of engineering an enzymehaving acetoacetyl-CoA substrate specificity and acetoacetyl-CoAspecific hydrolase activity, the method comprising: a) selecting anenzyme having acetoacetyl-CoA transferase activity, and b) substitutinga glutamic acid residue to an aspartic acid residue at a positioncorresponding to amino acid position 51 of SEQ ID NO: 1 in the enzymehaving acetoacetyl-CoA transferase activity to produce an engineeredenzyme.

In an embodiment of the disclosure, an active site glutamate residue atposition 51 of SEQ ID NO: 1, or an active site glutamic acid residue atposition 46 of SEQ ID NO: 3, or an active site glutamic acid residue atposition 333 of SEQ ID NO: 5, is substituted with an aspartate residueusing site direct mutagenesis to generate a CoA hydrolase (i.e., athioesterase) with higher activity on acetoacetyl-CoA versus acetyl-CoA.

Alternatively, in an embodiment of the disclosure an acetoacetyl-CoAhydrolase may be engineered from a transferase by directed evolution. Inan exemplary method, libraries of at least partially random mutatedacetoacetyl-CoA transferases are created and a mutant with the desiredhydrolase activity is identified through appropriate screening andselection methods (i.e. detection of free CoA after contacting enzymevariant with acetoacetyl-CoA, but without acceptor molecule). Such amethod can result in other mutations than an exchange of the activeglutamate acid residue to result in hydrolase activity. For instance,the three dimensional structure of the protein could get changed in sucha way, that the distance between substrate and acid group of the activeglutamic acid residue is increased to the same extent as in areplacement of the active glutamic acid with an aspartic acid residue,with similar effects on enzyme activity.

It will be appreciated by one of skill in the art that the active siteglutamate residue of an enzyme with acetoacetyl-CoA transferase activitycan be readily identified in any known transferase (see, Table 1) bysequence alignment of such enzyme with SEQ ID NO: 1 and that any knowntransferase can be modified to produce an acetoacetyl-CoA specifichydrolase. Such an alignment permits the identification of the glutamicacid residue at a position corresponding to amino acid position 51 ofSEQ ID NO: 1 to be substituted with an aspartic acid residue. It willalso be appreciated that not all transferases can accept acetoacetyl-CoAas a substrate. As such, those transferases that can acceptacetoacetyl-CoA as a substrate are preferred for use in the methods ofthe disclosure. Optionally, the engineered acetoacetyl-CoA specifichydrolase may be further modified by any methods known in the artincluding, by random mutagenesis, to increase hydrolase activity.

Exemplary enzymes suitable to accept acetoacetyl-CoA as substrate areset forth in Table 2 and are found among the subfamily of transferasesacting at 3-oxoacids (Table 1). These enzymes can be engineered to notconsume the equimolar amount of the acceptor acid molecule asco-substrate, but instead perform the hydrolysis of the thioester boundand liberate acetoacetate and free Coenzyme A (HCoA) as products. Withthe exception of Uniprot No. P37766 (this sequence is a fusion of analpha and beta subunit), Table 1 lists the beta subunit of aCoA-transferase. CoA-transferases are comprised of an alpha subunit anda beta subunit, and as such, those beta subunits listed in Table 1 mustbe combined with an alpha subunit in order to produce a catalyticallyactive CoA-transferase. It will be understood that the CoA-transferasebeta subunits listed in Table 1 may be combined with any knownCoA-transferase alpha subunit that renders the combination of the betasubunit and alpha subunit catalytically active.

TABLE 1 Exemplary 3-oxoacid CoA-transferases. Gene Uniprot EntryCatalytic Protein Name Name Organism Uniprot Name Glu Acetate CoA- atoAEscherichia P76459 ATOA_ECOLI E46 transferase subunit coli (strain betaK12) Acetate CoA- YdiF Escherichia P37766 YDIF_ECOLI E333 transferasecoli (strain K12) Acetate CoA- atoA Haemophilus P44874 ATOA_HAEIN E46transferase subunit influenzae beta 3-oxoadipate CoA- catJ PseudomonasQ8VPF2 CATJ_PSESB E51 transferase subunit sp. B Butyrate- ctfBClostridium P23673 CTFB_CLOAB E51 acetoacetate CoA- acetobutylicumtransferase subunit B Glutaconate CoA- gctB Acidaminococcus Q59112GCTB_ACIFV E54 transferase subunit fermentans B (strain ATCC 25085/ DSM20731/ VR4) 3-oxoadipate CoA- pcaJ Acinetobacter Q59091 PCAJ_ACIAD E50transferase subunit catJ sp. (strain ADP1) 3-oxoadipate CoA- pcaJPseudomonas P0A101 PCAJ_PSEPK E50 transferase subunit putida (strainKT2440) 3-oxoadipate CoA- pcaJ Pseudomonas P0A102 PCAJ_PSEPU E50transferase subunit putida (Arthrobacter siderocapsulatus) Probablesuccinyl- scoB Bacillus subtilis P42316 SCOB_BACSU E47 CoA:3-ketoacid(strain 168) coenzyme A Succinyl-CoA:3- scoB Helicobacter Q9ZLE4SCOB_HELPJ E43 ketoacid pylori (strain coenzyme A J99) transferase(Campylobacter pylori J99) Succinyl-CoA:3- scoB Helicobacter P56007SCOB_HELPY E43 ketoacid pylori (strain coenzyme A ATCC 700392/transferase 26695) (Campylobacter pylori) Probable succinyl- scoBMycobacterium P63651 SCOB_MYCBO E50 CoA:3-ketoacid bovis (straincoenzyme A ATCC BAA-935/ transferase AF2122/97) Probable succinyl- scoBMycobacterium P63650 SCOB_MYCTU E50 CoA:3-ketoacid tuberculosis coenzymeA transferase Succinyl-CoA:3- IpsJ Xanthomonas B0RVK3 SCOB_XANCB E47ketoacid campestris pv. coenzyme A campestris transferase (strain B100)Succinyl-CoA:3- IpsJ Xanthomonas P0C718 SCOB_XANCP E47 ketoacidcampestris pv. coenzyme A campestris transferase (strain ATCC33913/NCPPB 528/LMG 568) Putative CoA- Rv3552 Mycobacterium P63652Y3552_MYCTU E52 transferase subunit tuberculosis beta Rv3552 PutativeCoA- Mb3582 Mycobacterium P63653 Y3582_MYCBO E52 transferase subunitbovis (strain beta Mb3582 ATCC BAA-935/ AF2122/97) Probable yodRBacillus subtilis O34466 YODR_BACSU E50 coenzyme A (strain 168)transferase subunit beta

TABLE 2 Exemplary 3-oxoacid CoA-transferases able to acceptacetoacetyl-CoA as substrate. Gene Protein Names Names Organism UniprotEntry Name Acetate CoA- atoA Escherichia coli P76459 ATOA_ECOLItransferase (strain K12) subunit beta Acetate CoA- YdiF Escherichia coliP37766 YDIF_ECOLI transferase (strain K12) Butyrate-acetoacetate ctfBClostridium P23673 CTFB_CLOAB CoA-transferase acetobutylicum subunit BProbable succinyl- scoB Bacillus subtilis P42316 SCOB_BACSUCoA:3-ketoacid (strain 168) coenzyme A transferase

Modification of Microorganism

A microorganism may be modified (e.g., genetically engineered) by anymethod known in the art to comprise and/or express one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of a fermentable carbon source to one or more products. Suchmicroorganism may comprise a polynucleotide coding for an engineeredenzyme having acetoacetyl-CoA substrate specificity and acetoacetyl-CoAspecific hydrolase activity (e.g., an enzyme that comprises i) an aminoacid sequence of an enzyme having acetoacetyl-CoA transferase activityand ii) a substitution of a glutamic acid residue to an aspartic acidresidue at a position corresponding to amino acid position 51 of SEQ IDNO: 1).

Pathways that utilize an engineered enzyme having acetoacetyl-CoAsubstrate specificity and acetoacetyl-CoA specific hydrolase activityare shown below. Such pathways are merely exemplary and represent a fewof the ways in which the engineered enzyme disclosed herein may beexploited to catalyze the conversion of a fermentable carbon source toone or more desired end-products.

In some embodiments, a microorganism may be modified (e.g., geneticallyengineered) by any method known in the art to comprise and/or expressone or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of a fermentable carbon source to intermediates ina pathway for the co-production of 1-propanol and 2-propanol. Suchenzymes may include any of those enzymes as set forth in FIG. 4 or 5.For example, the microorganism may be modified to comprise one or morepolynucleotides coding for enzymes that catalyze a conversion ofdihydroxyacetone phosphate or pyruvate to 1-propanol.

In some embodiments, the microorganism may comprise one or moreexogenous polynucleotides encoding one or more enzymes in pathways forthe co-production of 1-propanol and 2-propanol from a fermentable carbonsource under anaerobic conditions.

In some embodiments, the microorganism may comprise one or morepolynucleotides coding for enzymes in a pathway that catalyzes aconversion of pyruvate to 2-propanol including, for example, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of pyruvate to acetyl-CoA, one or more polynucleotides codingfor enzymes in a pathway that catalyze a conversion of acetyl-CoA toacetoacetyl-CoA, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of acetoacetyl-CoA to acetoacetate,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of acetoacetate to acetone, and/or one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of acetone to 2-propanol. Enzymes catalyzing any of theseconversions may include, for example, those enzymes listed in Table 3.

In some embodiments, the non-naturally occurring microorganism maycomprise one or more polynucleotides coding for enzymes in a pathwaythat catalyzes a conversion of dihydroxyacetone-phosphate to 1-propanolincluding, for example: one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of dihydroxyacetone-phosphate tomethylglyoxal, one or more polynucleotides coding for enzymes in apathway that catalyze a conversion of methylglyoxal to hydroxyacetone,one or more polynucleotides coding for enzymes in a pathway thatcatalyze a conversion of hydroxyacetone to 1,2-propanediol, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of methylglyoxal to lactaldehyde, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion oflactaldehyde to 1,2-propanediol, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of 1,2-propanediol topropionaldehyde, and/or one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of propionaldehyde to1-propanol. Enzymes catalyzing any of these conversions may include, forexample, those enzymes listed in Table 4.

In some embodiments, the non-naturally occurring microorganism maycomprise one or more polynucleotides coding for enzymes in a pathwaythat catalyzes a conversion of lactate to 1-propanol including, forexample, one or more polynucleotides coding for enzymes in a pathwaythat catalyze a conversion of lactate to lactoyl-CoA, one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of lactoyl-CoA to lactaldehyde, one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion oflactaldehyde to 1,2-propanediol, one or more polynucleotides coding forenzymes in a pathway that catalyze a conversion of 1,2-propanediol topropionaldehyde, and/or one or more polynucleotides coding for enzymesin a pathway that catalyze a conversion of propionaldehyde to1-propanol. Enzymes catalyzing any of these conversions may include, forexample, those enzymes listed in Table 5.

A modified microorganism as provided herein may comprise:

-   -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of lactate to pyruvate,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of pyruvate to cytosolic acetyl-CoA,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of acetyl-CoA to acetoacetyl-CoA,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of acetoacetyl-CoA to AcAcetate,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of AcAcetate to acetone,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of acetone to 2-propanol,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of dihydroxyacetone phosphate to        methylglyoxal,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of methylglyoxal to lactaldehyde,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of methylglyoxal to hydroxyacetone,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of hydroxyacetone to 1,2-propanediol,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of lactaldehyde to 1,2-propanediol,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of 1,2-propanediol to propionaldehyde,        and/or    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of propionaldehyde to 1-propanol.        In some embodiments, the modified microorganism has a disruption        in each of the one or more polynucleotides that code for enzymes        that decarboxylate pyruvate and associated transcription factor        (e.g., pyruvate decarboxylase 1, 2, 5, and 6). In some        embodiments, the modified microorganism is capable of growth on        a C6 carbon source under anaerobic conditions. In some        embodiments, the modified microorganism has a disruption in each        of the one or more polynucleotides that code for enzymes that        decarboxylate pyruvate and associated transcription factor        (e.g., pyruvate decarboxylase 1, 2, 5, and 6) and is capable of        growth on a C6 carbon source under anaerobic conditions.

A modified microorganism as provided herein may comprise:

-   -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of pyruvate to lactate,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of lactate to lactoyl-CoA,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of lactoyl-CoA to lactaldehyde,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of lactate and acetyl-CoA to lactoyl-CoA        and acetic acid;    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of lactaldehyde to 1,2-propanediol,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of 1,2-propanediol to propionaldehyde,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of propionaldehyde to 1-propanol,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of pyruvate to acetyl-CoA,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of acetyl-CoA to acetoacetyl-CoA,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of acetoacetyl-CoA to AcAcetate,    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of AcAcetate to acetone, and/or    -   one or more polynucleotides coding for enzymes in a pathway that        catalyzes a conversion of acetone to 2-propanol.        In some embodiments, the modified microorganism has a disruption        in each of the one or more polynucleotides that code for enzymes        that decarboxylate pyruvate (e.g., pyruvate decarboxylase 1, 5,        and 6). In some embodiments, the modified microorganism is        capable of growth on a C6 carbon source under anaerobic        conditions. In some embodiments, the modified microorganism has        a disruption in each of the one or more polynucleotides that        code for enzymes that decarboxylate pyruvate (e.g., pyruvate        decarboxylase 1, 5, and 6) and is capable of growth on a C6        carbon source under anaerobic conditions.

Exemplary enzymes that convert a fermentable carbon source such asglucose to 1-propanol (Pathways B and C) and/or 2-propanol (Pathway A)including, enzyme substrates, and enzyme reaction products associatedwith the conversions are presented in Tables 3 to 5 below. The enzymereference identifier listed in Tables 3 to 5 correlates with the enzymenumbering used in FIGS. 4 and 5, which schematically represents theenzymatic conversion of a fermentable carbon source such as glucose todihydroxyacetone phosphate or lactate and pyruvate. Dihydroxyacetonephosphate or lactate and pyruvate may be further converted to 1-propanoland/or 2-propanol, using any combination of those enzymes provided inTables 3 to 5 above including, all of those enzymes as provided in Table3 to 5 below.

TABLE 3 Pathway A (2-propanol from pyruvate) Enzyme EC No. Enzyme nameNumber Reaction A1. Formate-C acetyltransferase 2.3.1.54 Pyruvate + CoA→ Formate-C acetyltransferase 1.97.1.4 Acetyl-CoA + formate activatingenzyme A2. Pyruvate dehydrogenase 1.2.4.1 Pyruvate + CoA + 2.3.1.12 NAD⁺→ Acetyl-CoA + 1.8.1.4 CO₂ + NADH B. Thiolase 2.3.1.9 2 acetyl-Coa →acetoacetyl-CoA + CoA C. Acetoacetyl-CoA 2.8.3.8 acetoacetyl-Coa +acetyltransferase acetate → acetoacetate + (engineered as describedacetyl-CoA herein) D. Acetatoacetate 4.1.1.4 acetoacetate →decarboxylase acetone + CO2 E. Secondary alcohol 1.1.1.2 acetone +NAD(P)H→ dehydrogenase 2-propanol + NAD(P)+

TABLE 4 Pathway B (1-propanol from Dihydroxyacetone phosphate Enzyme ECNo. Enzyme name Number Reaction F1. methylglyoxal 4.2.3.3dihydroxyacetone phosphate → synthase methylglyoxal F2. methylglyoxal4.2.3.3 dihydroxyacetone phosphate → synthase, phosphate methylglyoxalinsensitive G. Methylglyoxal 1.1.1.- Methylglyoxal → lactaldehydereductase H. Methylglyoxal 1.1.1.78 methylglyoxal → hydroxyacetonereductase I. methylglyoxal 1.1.1.- Hydroxyacetone + NAD(P)H + reductaseH⁺ → 1,2-propanediol + NAD(P)⁺ [multifunctional] J. methylglyoxal1.1.1.- Lactaldehyde + NAD(P)H + H⁺ → reductase 1,2-propanediol +NAD(P)⁺ [multifunctional] K. 1,2 propanediol 4.2.1.30 R/S 1,2propanediol → dehydratase proprionaldehyde L. 1-propanol 1.1.1.-proprionaldehyde + NADH → dehydrogenase propanol + NAD+

TABLE 5 Pathway C (1-propanol from lactate) Enzyme EC No. Enzyme nameNumber Reaction M1. D-Lactate 1.1.1.28 Pyruvate + NAD(P)H + H⁺ →dehydrogenase D-Lactate + NAD(P)⁺ M2. L-Lactate 1.1.1.27 Pyruvate +NAD(P)H + H⁺ → dehydrogenase L-Lactate + NAD(P)⁺ N. Propionate CoA-2.8.3.1 Lactate + Acetyl-CoA → lactoyl- transferase* CoA + _acetic acidO. Lactoyl-CoA 2.3.3.- Lactate + CoA + ATP → lactoyl- synthase CoA + AMPP. 1,2-propanediol 1.2.1.- Lactoyl-CoA + NAD(P)H + H⁺ → oxidoreductaseLactaldehyde + NAD(P)⁺ Q. Lactaldehyde 1.1.1.77 L-Lactaldehyde +NAD(P)H + H⁺ → reductase L-1,2-propanediol + NAD(P)⁺ J. methylglyoxal1.1.1.- Lactaldehyde + NAD(P)H + H⁺ → reductase 1,2-propanediol +NAD(P)⁺ [multifunctional] K. 1,2 propanediol 4.2.1.28 R/S 1,2propanediol → dehydratase propionaldehyde L. 1-propanol 1.1.1.-Propionaldehyde → 1-propanol dehydrogenase *enzyme with homologousfunction but altered substrate specificity is required/preferred

The microorganism may be an archea, bacteria, or eukaryote. In someembodiments, the bacteria is a Propionibacterium, Propionispira,Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillusincluding, for example, Pelobacter propionicus, Clostridium propionicum,Clostridium acetobutylicum, Lactobacillus, Propionibacteriumacidipropionici or Propionibacterium freudenreichii. In someembodiments, the eukaryote is a yeast, filamentous fungi, protozoa, oralgae. In some embodiments, the yeast is Saccharomyces cerevisiae,Kluyveromyces lactis or Pichia pastoris.

In some embodiments, the microorganism is additionally modified tocomprise one or more tolerance mechanisms including, for example,tolerance to a produced molecule (i.e., methylglyoxal, 1-propanol,2-propanol, or butadiene), and/or organic solvents. A microorganismmodified to comprise such a tolerance mechanism may provide a means toincrease titers of fermentations and/or may control contamination in anindustrial scale process.

The present disclosure also provides microorganisms (e.g., S.cerevisiae) for the co-production of 2-propanol and 1-propanol and/or1,2-propanediol. Microorganisms may be modified so that they mayco-produce 2-propanol and 1-propanol and/or 1,2-propanediol. In anembodiment, a microorganism may have its native ethanol productionreduced or eliminated (i.e., shut off). In an embodiment, to eliminateethanol production in the microorganism the activity of pyruvatedecarboxylase (i.e., the enzyme which decarboxylates pyruvate and in theprocess makes acetaldehyde and CO₂) may be disrupted including, forexample, knocked-out. Pyruvate decarboxylase comes in three isoforms inyeast and its activity can be mostly knocked out by deleting the genesPDC1, PDC5, and PDC6. Without wishing to be bound by a theory of theinvention, the elimination of the pyruvate decarboxylase activity in thecell's cytoplasm renders the yeast cell unable to grow under anaerobicconditions due to two factors: (1) the lack of an alternative route forcytoplasmic acetyl-CoA production, due to the lack of acetaldehyde thatwould be converted to acetate and acetyl-coA; and (2) a redox imbalancedue to excess NADH because the NADH is no longer oxidized in theconversion of acetaldehyde to ethanol. Thus, it is necessary to alsoalter the ability of the microorgansim to import glucose by truncating atranscription factor of the glucose importer called MTH1. Thistruncation then restores the ability of the ΔPDC1,5,6 mutantmicroorganism to survive on C6 sugars. In an embodiment, one or morepolynucleotides coding for a bacterial pyruvate formate lyase orcytosolic pyruvate dehydrogenase complex may be inserted into themicroorganism to convert pyruvate into Acetyl CoA in the cytosol. In anembodiment, the microorganism may be modified to comprise one or morepolynucleotides that code for enzymes in a pathway for the coproductionof 2-propanol and 1-propanol and/or 1,2-propanediol. In a furtherembodiment, the microorganism may be modified to comprise anacetoacetylCoA hydrolase. Such an acetoacetylCoA hydrolase may beengineered from an acetoacetylCoA:acetate transferase by making a singleGlu-Asp mutation in the acetoacetylCoA:acetate transferase. In anadditional embodiment, a microorganism may be modified to comprise oneor more polynucleotides coding for a B12-independent dehydratase fromthe organism Roseburia inuvolurans to convert 1,2-propanediol topropanaldehyde. Microorganims that comprise one or more of themodifications set forth above are termed a non-naturally occuringmicroroganism or a modified microorganism.

WO2004099425 discloses the overproduction of pyruvate in S. cerevisiaeby knocking out pyruvate decarboxylase activity and a directed evolutionprocess that allowed this triple mutant to grow on glucose due to atruncation of the MTH1 transcription factor. However, the scope stoppedat the overproduction of pyruvate in aerobic fermentation systems. Theuse of oxygen, in this context, was essential as there is a huge buildupof NADH in the cell due to the fact that NADH is no longer oxidized inthe conversion of acetaldehyde to ethanol.

The present disclosure also provides modified microorganisms thatcomprise: a disruption of one or more enzymes that decarboxylatepyruvate and/or a disruption of one or more transcription factors of oneor more enzymes that decarboxylate pyruvate; a genetic modification thatsubstantially decreases glucose import into the microorganism; one ormore polynucleotides encoding an acetoacetyl-CoA specific hydrolase asdisclosed herein, one or more polynucleotides encoding one or moreenzymes in a pathway that produces cytosolic acetyl-CoA; one or morepolynucleotides encoding one or more enzymes in a pathway that catalyzea conversion of cytosolic acetyl-CoA to 2-propanol; and one or morepolynucleotides encoding one or more enzymes in a pathway that catalyzea conversion of dihydroxyacetone-phosphate to 1-propanol and/or1,2-propanediol.

The present disclosure further comprises a pyruvate overproducing cellable to produce cytosolic Acetyl-CoA inserting for example, bacterialpyruvate formate lyase or cytosolic pyruvate dehydrogenase complex toconvert pyruvate into Acetyl-CoA in the cytosol of the eukaryote cell.The insertion of pyruvate formate lyase in to a PDC-negative yeaststrain was disclosed by Waks and Silver in Engineering a SyntheticDual-Organism System for Hydrogen Production (Applied and EnvironmentalMicrobiology, vol. 75, n. 7, 2009, p. 1867-1875) without success inanaerobic growth or metabolism. Furthermore, the present disclosurefurther comprises a pyruvate overproducing cell able to producecytosolic Acetyl-CoA and to grow under anaerobic conditions by providinga temporary redox sink that allows reoxidation of NADH by introducing agene coding for a bacterial soluble NAD(P)+ transhydrogenase(Si-specific) (udhA gene from E. coli, E.C. number 1.6.1.1.) thatcatalyzes the interconversion of NADP++NADH=NADPH+NAD+. The concomitantexpression of the PFL and udhA enzymes to restore anaerobic growth tothe PDC-null yeast strain expressing the truncated MTH1 constitutes thefirst report of anaerobic growth of a PDC-null yeast strain and servesas a new eukaryotic chassis for the production of commodity chemicals.

In some embodiments, the disclosure contemplates the modification (e.g.,engineering) of one or more of the enzymes provided herein. Suchmodification may be performed to redesign the substrate specificity ofthe enzyme and/or to modify (e.g., reduce) its activity against otherssubstrates in order to increase its selectivity for a given substrate.Additionally or alternatively, one or more enzymes as provided hereinmay be engineered to alter (e.g., enhance including, for example,increase its catalytic activity or its substrate specificity) one ormore of its properties, including acceptance of different cofactors suchas NADH instead of NADPH.

In some embodiments, sequence alignment and comparative modeling ofproteins may be used to alter one or more of the enzymes disclosedherein. Homology modeling or comparative modeling refers to building anatomic-resolution model of the desired protein from its primary aminoacid sequence and an experimental three-dimensional structure of asimilar protein. This model may allow for the enzyme substrate bindingsite to be defined, and the identification of specific amino acidpositions that may be replaced to other natural amino acid in order toredesign its substrate specificity.

Variants or sequences having substantial identity or homology with thepolynucleotides encoding enzymes as disclosed herein may be utilized inthe practice of the disclosure. Such sequences can be referred to asvariants or modified sequences. That is, a polynucleotide sequence maybe modified yet still retain the ability to encode a polypeptideexhibiting the desired activity. Such variants or modified sequences arethus equivalents in the sense that they retain their intended function.Generally, the variant or modified sequence may comprise at least about40%-60%, preferably about 60%-80%, more preferably about 80%-90%, andeven more preferably about 90%-95% sequence identity with the nativesequence.

In some embodiments, a microorganism may be modified to expressincluding, for example, overexpress, one or more enzymes as providedherein. The microorganism may be modified by genetic engineeringtechniques (i.e., recombinant technology), classical microbiologicaltechniques, or a combination of such techniques and can also includenaturally occurring genetic variants to produce a genetically modifiedmicroorganism. Some of such techniques are generally disclosed, forexample, in Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Labs Press; and Selifonova et al. (2001)Appl. Environ. Microbiol. 67(8):3645).

A genetically modified microorganism may include a microorganism inwhich a polynucleotide has been inserted, deleted or modified (i.e.,mutated; e.g., by insertion, deletion, substitution, and/or inversion ofnucleotides), in such a manner that such modifications provide thedesired effect of expression (e.g., over-expression) of one or moreenzymes as provided herein within the microorganism. Geneticmodifications which result in an increase in gene expression or functioncan be referred to as amplification, overproduction, overexpression,activation, enhancement, addition, or up-regulation of a gene. Additionof cloned genes to increase gene expression can include maintaining thecloned gene(s) on replicating plasmids or integrating the cloned gene(s)into the genome of the production organism. Furthermore, increasing theexpression of desired cloned genes can include operatively linking thecloned gene(s) to native or heterologous transcriptional controlelements.

Where desired, the expression of one or more of the enzymes providedherein are under the control of a regulatory sequence that controlsdirectly or indirectly the expression of the enzyme in a time-dependentfashion during a fermentation reaction.

In some embodiments, a microorganism is transformed or transfected witha genetic vehicle such as, an expression vector comprising an exogenouspolynucleotide sequence coding for the enzymes provided herein.

Polynucleotide constructs prepared for introduction into a prokaryoticor eukaryotic host may typically, but not always, comprise a replicationsystem (i.e. vector) recognized by the host, including the intendedpolynucleotide fragment encoding the desired polypeptide, and maypreferably, but not necessarily, also include transcription andtranslational initiation regulatory sequences operably linked to thepolypeptide-encoding segment. Expression systems (expression vectors)may include, for example, an origin of replication or autonomouslyreplicating sequence (ARS) and expression control sequences, a promoter,an enhancer and necessary processing information sites, such asribosome-binding sites, RNA splice sites, polyadenylation sites,transcriptional terminator sequences, mRNA stabilizing sequences,nucleotide sequences homologous to host chromosomal DNA, and/or amultiple cloning site. Signal peptides may also be included whereappropriate, preferably from secreted polypeptides of the same orrelated species, which allow the protein to cross and/or lodge in cellmembranes or be secreted from the cell.

The vectors can be constructed using standard methods (see, e.g.,Sambrook et al., Molecular Biology: A Laboratory Manual, Cold SpringHarbor, N. Y. 1989; and Ausubel, et al., Current Protocols in MolecularBiology, Greene Publishing, Co. N.Y, 1995).

The manipulation of polynucleotides of the present disclosure includingpolynucleotides coding for one or more of the enzymes disclosed hereinis typically carried out in recombinant vectors. Numerous vectors arepublicly available, including bacterial plasmids, bacteriophage,artificial chromosomes, episomal vectors and gene expression vectors,which can all be employed. A vector of use according to the disclosuremay be selected to accommodate a protein coding sequence of a desiredsize. A suitable host cell is transformed with the vector after in vitrocloning manipulations. Host cells may be prokaryotic, such as any of anumber of bacterial strains, or may be eukaryotic, such as yeast orother fungal cells, insect or amphibian cells, or mammalian cellsincluding, for example, rodent, simian or human cells. Each vectorcontains various functional components, which generally include acloning site, an origin of replication and at least one selectablemarker gene. If given vector is an expression vector, it additionallypossesses one or more of the following: enhancer element, promoter,transcription termination and signal sequences, each positioned in thevicinity of the cloning site, such that they are operatively linked tothe gene encoding a polypeptide repertoire member according to thedisclosure.

Vectors, including cloning and expression vectors, may contain nucleicacid sequences that enable the vector to replicate in one or moreselected host cells. For example, the sequence may be one that enablesthe vector to replicate independently of the host chromosomal DNA andmay include origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeast and viruses. For example, the origin of replication from theplasmid pBR322 is suitable for most Gram-negative bacteria, the 2 micronplasmid origin is suitable for yeast, and various viral origins (e.g. SV40, adenovirus) are useful for cloning vectors in mammalian cells.Generally, the origin of replication is not needed for mammalianexpression vectors unless these are used in mammalian cells able toreplicate high levels of DNA, such as COS cells.

A cloning or expression vector may contain a selection gene alsoreferred to as a selectable marker. This gene encodes a proteinnecessary for the survival or growth of transformed host cells grown ina selective culture medium. Host cells not transformed with the vectorcontaining the selection gene will therefore not survive in the culturemedium. Typical selection genes encode proteins that confer resistanceto antibiotics and other toxins, e.g. ampicillin, neomycin,methotrexate, hygromycin, thiostrepton, apramycin or tetracycline,complement auxotrophic deficiencies, or supply critical nutrients notavailable in the growth media.

The replication of vectors may be performed in E. coli (e.g., strain TB1or TG1, DH5α, DH10β, JM110). An E. coli-selectable marker, for example,the β-lactamase gene that confers resistance to the antibioticampicillin, may be of use. These selectable markers can be obtained fromE. coli plasmids, such as pBR322 or a pUC plasmid such as pUC18 orpUC19, or pUC119.

Expression vectors may contain a promoter that is recognized by the hostorganism. The promoter may be operably linked to a coding sequence ofinterest. Such a promoter may be inducible or constitutive.Polynucleotides are operably linked when the polynucleotides are in arelationship permitting them to function in their intended manner.

Promoters suitable for use with prokaryotic hosts may include, forexample, the α-lactamase and lactose promoter systems, alkalinephosphatase, the tryptophan (trp) promoter system, the erythromycinpromoter, apramycin promoter, hygromycin promoter, methylenomycinpromoter and hybrid promoters such as the tac promoter. Moreover, hostconstitutive or inducible promoters may be used. Promoters for use inbacterial systems will also generally contain a Shine-Dalgarno sequenceoperably linked to the coding sequence.

Viral promoters obtained from the genomes of viruses include promotersfrom polyoma virus, fowlpox virus, adenovirus (e.g., Adenovirus 2 or 5),herpes simplex virus (thymidine kinase promoter), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus (e.g., MoMLV,or RSV LTR), Hepatiti B virus, Myeloproliferative sarcoma virus promoter(MPSV), VISNA, and Simian Virus 40 (SV40). Heterologous mammalianpromoters include, e.g., the actin promoter, immunoglobulin promoter,heat-shock protein promoters.

The early and late promoters of the SV40 virus are conveniently obtainedas a restriction fragment that also contains the SV40 viral origin ofreplication (see, e.g., Fiers et al., Nature, 273:113 (1978); Mulliganand Berg, Science, 209:1422-1427 (1980); and Pavlakis et al., Proc.Natl. Acad. Sci. USA, 78:7398-7402 (1981)). The immediate early promoterof the human cytomegalovirus (CMV) is conveniently obtained as a HindIII E restriction fragment (see, e.g., Greenaway et al., Gene,18:355-360 (1982)). A broad host range promoter, such as the SV40 earlypromoter or the Rous sarcoma virus LTR, is suitable for use in thepresent expression vectors.

Generally, a strong promoter may be employed to provide for high leveltranscription and expression of the desired product. Among theeukaryotic promoters that have been identified as strong promoters forhigh-level expression are the SV40 early promoter, adenovirus major latepromoter, mouse metallothionein-I promoter, Rous sarcoma virus longterminal repeat, and human cytomegalovirus immediate early promoter (CMVor CMV IE). In an embodiment, the promoter is a SV40 or a CMV earlypromoter.

The promoters employed may be constitutive or regulatable, e.g.,inducible. Exemplary inducible promoters include jun, fos andmetallothionein and heat shock promoters. One or both promoters of thetranscription units can be an inducible promoter. In an embodiment, theGFP is expressed from a constitutive promoter while an induciblepromoter drives transcription of the gene coding for one or more enzymesas disclosed herein and/or the amplifiable selectable marker.

The transcriptional regulatory region in higher eukaryotes may comprisean enhancer sequence. Many enhancer sequences from mammalian genes areknown e.g., from globin, elastase, albumin, α-fetoprotein and insulingenes. A suitable enhancer is an enhancer from a eukaryotic cell virus.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the enhancer of the cytomegalovirus immediate earlypromoter (Boshart et al. Cell 41:521 (1985)), the polyoma enhancer onthe late side of the replication origin, and adenovirus enhancers (seealso, e.g., Yaniv, Nature, 297:17-18 (1982) on enhancing elements foractivation of eukaryotic promoters). The enhancer sequences may beintroduced into the vector at a position 5′ or 3′ to the gene ofinterest, but is preferably located at a site 5′ to the promoter.

Yeast and mammalian expression vectors may contain prokaryotic sequencesthat facilitate the propagation of the vector in bacteria. Therefore,the vector may have other components such as an origin of replication(e.g., a nucleic acid sequence that enables the vector to replicate inone or more selected host cells), antibiotic resistance genes forselection in bacteria, and/or an amber stop codon which can permittranslation to read through the codon. Additional eukaryotic selectablegene(s) may be incorporated. Generally, in cloning vectors the origin ofreplication is one that enables the vector to replicate independently ofthe host chromosomal DNA, and includes origins of replication orautonomously replicating sequences. Such sequences are well known, e.g.,the ColE1 origin of replication in bacteria. Various viral origins(e.g., SV40, polyoma, adenovirus, VSV or BPV) are useful for cloningvectors in mammalian cells. Generally, a eukaryotic replicon is notneeded for expression in mammalian cells unless extrachromosomal(episomal) replication is intended (e.g., the SV40 origin may typicallybe used only because it contains the early promoter).

To facilitate insertion and expression of different genes coding for theenzymes as disclosed herein from the constructs and expression vectors,the constructs may be designed with at least one cloning site forinsertion of any gene coding for any enzyme disclosed herein. Thecloning site may be a multiple cloning site, e.g., containing multiplerestriction sites.

The plasmids may be propagated in bacterial host cells to prepare DNAstocks for subcloning steps or for introduction into eukaryotic hostcells. Transfection of eukaryotic host cells can be any performed by anymethod well known in the art. Transfection methods include lipofection,electroporation, calcium phosphate co-precipitation, rubidium chlorideor polycation mediated transfection, protoplast fusion andmicroinjection. Preferably, the transfection is a stable transfection.The transfection method that provides optimal transfection frequency andexpression of the construct in the particular host cell line and type,is favored. Suitable methods can be determined by routine procedures.For stable transfectants, the constructs are integrated so as to bestably maintained within the host chromosome.

Vectors may be introduced to selected host cells by any of a number ofsuitable methods known to those skilled in the art. For example, vectorconstructs may be introduced to appropriate cells by any of a number oftransformation methods for plasmid vectors. For example, standardcalcium-chloride-mediated bacterial transformation is still commonlyused to introduce naked DNA to bacteria (see, e.g., Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), but electroporation andconjugation may also be used (see, e.g., Ausubel et al., 1988, CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.).

For the introduction of vector constructs to yeast or other fungalcells, chemical transformation methods may be used (e.g., Rose et al.,1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.). Transformed cells may be isolated onselective media appropriate to the selectable marker used.Alternatively, or in addition, plates or filters lifted from plates maybe scanned for GFP fluorescence to identify transformed clones.

For the introduction of vectors comprising differentially expressedsequences to mammalian cells, the method used may depend upon the formof the vector. Plasmid vectors may be introduced by any of a number oftransfection methods, including, for example, lipid-mediatedtransfection (“lipofection”), DEAE-dextran-mediated transfection,electroporation or calcium phosphate precipitation (see, e.g., Ausubelet al., 1988, Current Protocols in Molecular Biology, John Wiley & Sons,Inc., NY, N.Y.).

Lipofection reagents and methods suitable for transient transfection ofa wide variety of transformed and non-transformed or primary cells arewidely available, making lipofection an attractive method of introducingconstructs to eukaryotic, and particularly mammalian cells in culture.For example, LipofectAMINE™ (Life Technologies) or LipoTaxi™(Stratagene) kits are available. Other companies offering reagents andmethods for lipofection include Bio-Rad Laboratories, CLONTECH, GlenResearch, InVitrogen, JBL Scientific, MBI Fermentas, PanVera, Promega,Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

The host cell may be capable of expressing the construct encoding thedesired protein, processing the protein and transporting a secretedprotein to the cell surface for secretion. Processing includes co- andpost-translational modification such as leader peptide cleavage, GPIattachment, glycosylation, ubiquitination, and disulfide bond formation.Immortalized host cell cultures amenable to transfection and in vitrocell culture and of the kind typically employed in genetic engineeringare preferred. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (CO 7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 derivatives adapted for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977); babyhamster kidney cells (BHK, ATCC CCL 10); DHFR-Chinese hamster ovarycells (ATCC CRL-9096); dp12.CHO cells, a derivative of CHO/DHFR-(EP307,247 published 15 Mar. 1989); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci., 383:44-68 (1982)); PEER human acutelymphoblastic cell line (Ravid et al. Int. J. Cancer 25:705-710 (1980));MRC 5 cells; FS4 cells; human hepatoma line (Hep G2), human HT1080cells, KB cells, JW-2 cells, Detroit 6 cells, NIH-3T3 cells, hybridomaand myeloma cells. Embryonic cells used for generating transgenicanimals are also suitable (e.g., zygotes and embryonic stem cells).

Suitable host cells for cloning or expressing polynucleotides (e.g.,DNA) in vectors may include, for example, prokaryote, yeast, or highereukaryote cells. Suitable prokaryotes for this purpose includeeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis (e.g., B.licheniformis 41 P disclosed in DD 266,710 published Apr. 12, 1989),Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E.coli cloning host is E. coli 294 (ATCC 31,446), although other strainssuch as E. coli B, E. coli X1776 (ATCC 31,537), E. coli JM110 (ATCC47,013) and E. coli W3110 (ATCC 27,325) are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast may be suitable cloning or expression hosts for vectorscomprising polynucleotides coding for one or more enzymes. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms. However, a number of other genera,species, and strains are commonly available and useful herein, such asSchizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis,K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii(ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906),K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichiapastors (EP 183,070); Candida; Trichoderma reesia (EP 244,234);Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis;and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

When the enzyme is glycosylated, suitable host cells for expression maybe derived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyxmori (silk moth) have been identified. A variety of viral strains fortransfection are publicly available, e.g., the L-1 variant of Autographacalifornica NPV and the Bm-5 strain of Bombyx mori NPV, and such virusesmay be used as the virus herein according to the present disclosure,particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,tobacco, lemna, and other plant cells can also be utilized as hostcells.

Examples of useful mammalian host cells are Chinese hamster ovary cells,including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamsterovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); monkey kidney CV1 line transformed by SV40 (CO 7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, (Graham et al., J. Gen Virol. 36: 59,1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells(TM4, Mather, (Biol. Reprod. 23: 243-251, 1980); monkey kidney cells(CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells(Mather et al., Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells;FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed or transfected with the above-describedexpression or cloning vectors for production of one or more enzymes asdisclosed herein or with polynucleotides coding for one or more enzymesas disclosed herein and cultured in conventional nutrient media modifiedas appropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences.

Host cells containing desired nucleic acid sequences coding for thedisclosed enzymes may be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle'sMedium ((DMEM), Sigma) are suitable for culturing the host cells. Inaddition, any of the media described in Ham et al., Meth. Enz. 58: 44,(1979); Barnes et al., Anal. Biochem. 102: 255 (1980); U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO87/00195; or U.S. Pat. Re. No. 30,985 may be used as culture media forthe host cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleotides (such asadeNOSine and thymidine), antibiotics (such as GENTAMYCIN™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

Any of the intermediates produced in any of the enzymatic pathwaysdisclosed herein may be an intermediate in the classical sense of theword in that they may be enzymatically converted to another intermediateor an end product. Alternatively, the intermediates themselves may beconsidered an end product.

Polynucleotides and Encoded Enzymes

Any known polynucleotide (e.g., gene) that codes for an enzyme orvariant thereof that is capable of catalyzing an enzymatic conversionincluding, for example, an enzyme as set forth in any one of Tables 3-5or FIGS. 4-5, is contemplated for use by the present disclosure. Suchpolynucleotides may be modified (e.g., genetically engineered) tomodulate (e.g., increase or decrease) the substrate specificity of anencoded enzyme, or the polynucleotides may be modified to change thesubstrate specificity of the encoded enzyme (e.g., a polynucleotide thatcodes for an enzyme with specificity for a substrate may be modifiedsuch that the enzyme has specificity for an alternative substrate).Preferred microorganisms may comprise polynucleotides coding for one ormore of the enzymes as set forth in Tables 3-5 and FIGS. 4-5.

Enzymes for catalyzing the conversions set forth in pathways A, B, and Cof Tables 3-5 and FIGS. 4-5 are categorized in Table 4 below.

TABLE 4 Exemplary Gene Identifier (GI) numbers Uniprot SEQ PathwaysFIGS. Enzyme No. EC No. Enzyme candidate Gene ID (aa) ID NO. A 4, 5 A2.3.1.54/ Formate-C PFLB P75793 7 1.97.1.4 acetyltransferase A 4, 5 A2.3.1.54/ Formate-C PFLA C4ZXZ6 8 1.97.1.4 acetyltransferase (activatingenzyme) A 4, 5 A 2.3.1.54/ Formate-C PFLB K9LI23 9 1.97.1.4acetyltransferase A 4, 5 A 2.3.1.54/ Formate-C PFLA Q6RFH6 10 1.97.1.4acetyltransferase (activating enzyme) A 4, 5 A 1.2.4.1/ Pyruvate pda1P16387 11 2.3.1.12/ dehydrogenase complex 1.8.1.4 A 4, 5 A 1.2.4.1/Pyruvate pdb1 P32473 12 2.3.1.12/ dehydrogenase complex 1.8.1.4 A 4, 5 A1.2.4.1/ Pyruvate lat1 P12695 13 2.3.1.12/ dehydrogenase complex 1.8.1.4A 4, 5 A 1.2.4.1/ Pyruvate lpd1 P09624 14 2.3.1.12/ dehydrogenasecomplex 1.8.1.4 A 4, 5 A 1.2.4.1/ Pyruvate pdx1 P16451 15 2.3.1.12/dehydrogenase complex 1.8.1.4 A 4, 5 A 1.2.4.1/ Pyruvate pdhA F2MRX7 162.3.1.12/ dehydrogenase complex 1.8.1.4 (E1 aplha) A 4, 5 B 3.1.2.—Acetoacetyl-CoA — — SEQ ID hydrolase NO: 2, 4, or 6 A 4, 5 D 4.1.1.4acetoacetate adc P23670 17 decarboxylase A 4, 5 D 4.1.1.4 acetoacetateadc A6M020 18 decarboxylase A 4, 5 E 1.1.1.2 secondary alcohol adhP25984 19 dehydrogenase B 4 F 4.2.3.3 methylglyoxal synthase mgsA P4298020 B 4 F 4.2.3.3 methylglyoxal synthase mgsA P0A731 21 B 4 F 4.2.3.3methylglyoxal synthase mgsA* P0A731 22 B 4 G 1.1.1.— methylglyoxalreductase, ydjg P77256 23 multifunctional B 4 H 1.1.1.78 methylglyoxalreductase ypr1 C7GMG9 24 B 4 I 1.1.1.304 methylglyoxal reductase, budCQ48436 25 multifunctional B, C 4, 5 J 1.1.1.77 lactaldehyde reductasefucO P0A9S1 26 B, C 4, 5 J 1.1.1.— methylglyoxal reductase yafB P3086327 [multifunctional] B, C 4, 5 K 4.2.1.30 glycerol dehydratase dhaB1Q8GEZ8 28 B, C 4, 5 K 4.2.1.30 glycerol dehydratase dhaB2 Q8GEZ7 29activator B, C 4, 5 K 4.2.1.30 diol dehydratase b1 Q1A666 30 B, C 4, 5 K4.2.1.30 diol dehydratase b2 Q1A665 31 activator B, C 4, 5 L 1.1.1.1alcohol dehydrogenase adh C6PZV5 32 C 5 M 1.1.1.28 D-Lactate ldhA P5264333 dehydrogenase C 5 M 1.1.1.27 L-Lactate ldhL2 P59390 34 dehydrogenaseC 5 M 1.1.1.27 L-lactate ldh2 P19858 35 dehydrogenase C 5 N 2.8.3.1propionate CoA- pct Q9L3F7 36 transferase* C 5 O 2.3.3.— Lactoyl-CoASynthase ACS1 Q01574 37 C 5 P 1.2.1.— CoA-dependent pduP Q9XDN1 38propionaldehyde dehydrogenase* C 5 Q 1.1.1.77 L-1,2-propanediol fucOP0A9S1 39 oxidoreductase

Methods for the Co-Production of 1-Propanol and 2-Propanol

1-propanol and 2-propanol may be produced by contacting any of thegenetically modified microorganisms provided herein with a fermentablecarbon source. Such methods may preferably comprise contacting afermentable carbon source with a microorganism comprising one or morepolynucleotides coding for enzymes in a pathway that catalyzes aconversion of the fermentable carbon source to any of the intermediatesprovided in FIGS. 4-5 (Tables 3-5) and one or more polynucleotidescoding for enzymes in a pathway that catalyze a conversion of the one ormore intermediates provided in FIGS. 4-5 (tables 3-5) to 1-propanol and2-propanol in a fermentation media; and expressing the one or morepolynucleotides coding for the enzymes in the pathway that catalyzes aconversion of the fermentable carbon source to the one or moreintermediates provided in FIGS. 4-5 (tables 3-5) and one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of the one or more intermediates provided in FIGS. 4-5(tables 3-5) to 1-propanol and 2-propanol.

The metabolic pathways that lead to the production of industriallyimportant compounds involve oxidation-reduction (redox) reactions. Forexample, during fermentation, glucose is oxidized in a series ofenzymatic reactions into smaller molecules with the concomitant releaseof energy. The electrons released are transferred from one reaction toanother through universal electron carriers, such Nicotinamide AdenineDinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate(NAD(P)), which act as cofactors for oxidoreductase enzymes. Inmicrobial catabolism, glucose is oxidized by enzymes using the oxidizedform of the cofactors (NAD(P)+ and/or NAD+) as cofactor thus generatingreducing equivalents in the form of the reduced cofactor (NAD(P)H andNADH). In order for fermentation to continue, redox-balanced metabolismis required, i.e., the cofactors must be regenerated by the reduction ofmicrobial cell metabolic compounds.

Microorganism-catalyzed fermentation for the production of naturalproducts is a widely known application of biocatalysis. Industrialmicroorganisms can affect multistep conversions of renewable feedstocksto high value chemical products in a single reactor. Products ofmicroorganism-catalyzed fermentation processes range from chemicals suchas ethanol, lactic acid, amino acids and vitamins, to high value smallmolecule pharmaceuticals, protein pharmaceuticals, and industrialenzymes. In many of these processes, the biocatalysts are whole-cellmicroorganisms, including microorganisms that have been geneticallymodified to express heterologous genes.

Some key parameters for efficient microorganism-catalyzed fermentationprocesses include the ability to grow microorganisms to a greater celldensity, increased yield of desired products, increased amount ofvolumetric productivity, removal of unwanted co-metabolites, improvedutilization of inexpensive carbon and nitrogen sources, adaptation tovarying fermenter conditions, increased production of a primarymetabolite, increased production of a secondary metabolite, increasedtolerance to acidic conditions, increased tolerance to basic conditions,increased tolerance to organic solvents, increased tolerance to highsalt conditions and increased tolerance to high or low temperatures.Inefficiencies in any of these parameters can result in highmanufacturing costs, inability to capture or maintain market share,and/or failure to bring fermented end-products to market.

The methods and compositions of the present disclosure can be adapted toconventional fermentation bioreactors (e.g., batch, fed-batch, cellrecycle, and continuous fermentation).

In some embodiments, a microorganism (e.g., a genetically modifiedmicroorganism) as provided herein is cultivated in liquid fermentationmedia (i.e., a submerged culture) which leads to excretion of thefermented product(s) into the fermentation media. In one embodiment, thefermented end product(s) can be isolated from the fermentation mediausing any suitable method known in the art.

In some embodiments, formation of the fermented product occurs during aninitial, fast growth period of the microorganism. In one embodiment,formation of the fermented product occurs during a second period inwhich the culture is maintained in a slow-growing or non-growing state.In one embodiment, formation of the fermented product occurs during morethan one growth period of the microorganism. In such embodiments, theamount of fermented product formed per unit of time is generally afunction of the metabolic activity of the microorganism, thephysiological culture conditions (e.g., pH, temperature, mediumcomposition), and the amount of microorganisms present in thefermentation process.

In some embodiments, the fermentation product is recovered from theperiplasm or culture medium as a secreted metabolite. In one embodiment,the fermentation product is extracted from the microorganism, forexample when the microorganism lacks a secretory signal corresponding tothe fermentation product. In one embodiment, the microorganisms areruptured and the culture medium or lysate is centrifuged to removeparticulate cell debris. The membrane and soluble protein fractions maythen be separated if necessary. The fermentation product of interest maythen be purified from the remaining supernatant solution or suspensionby, for example, distillation, fractionation, chromatography,precipitation, filtration, and the like.

The methods of the present disclosure are preferably preformed underanaerobic conditions. Both the degree of reduction of a product as wellas the ATP requirement of its synthesis determines whether a productionprocess is able to proceed aerobically or anaerobically. To produce1-propanol and 2-propanol or 1-propanol and butadiene via anaerobicmicrobial conversion, or at least by using a process with reduced oxygenconsumption, redox imbalances should be avoided. Several types ofmetabolic conversion steps involve redox reactions. Such redox reactionsinvolve electron transfer mediated by the participation of redoxcofactors such as NADH, NADPH and ferredoxin. Since the amounts of redoxcofactors in the cell are limited to permit the continuation ofmetabolic processes, the cofactors have to be regenerated. In order toavoid such redox imbalances, alternative ways of cofactor regenerationmay be engineered, and in some cases additional sources of ATPgeneration may be provided. Alternatively, oxidation and reductionprocesses may be separated spatially in bioelectrochemical systems(Rabaey and. Rozendal, 2010, Nature reviews, Microbiology, vol 8:706-716).

In some embodiment, redox imbalances may be avoided by using substrates(e.g., fermentable carbon sources) that are more oxidized or morereduced. for example, if the utilization of a substrate results in adeficit or surplus of electrons, a requirement for oxygen can becircumvented by using substrates that are more reduced or oxidized,respectively. For example, glycerol which is a major byproduct ofbiodiesel production is more reduced than sugars, and is therefore moresuitable for the synthesis of compounds whose production from sugarresults in cofactor oxidation, such as succinic acid. In someembodiments, if the conversion of a substrate to a product results in anelectron deficit, co-substrates can be added that function as electrondonors (Babel 2009, Eng. Life Sci. 9, 285-290). An important criterionfor the anaerobic use of co-substrates is that their redox potential ishigher than that of NADH (Geertman et al., 2006, FEMS Yeast Res. 6,1193-1203). If the conversion of substrate to produce results in anelectron surplus, co-substrates can be added that function as electronacceptors.

Methods for the Production of Polypropylene

1-propanol produced via methods disclosed herein may be dehydrated toform propylene, which may then be polymerized to produce polypropylenein a cost-effective manner.

Propylene is a chemical compound that is widely used to synthesize awide range of petrochemical products. For instance, this olefin is theraw material used for the production of polypropylene, its copolymersand other chemicals such as acrylonitrile, acrylic acid, epichloridrineand acetone. Propylene demand is growing faster than ethylene demand,mainly due to the growth of market demand for polypropylene. Propyleneis polymerized to produce thermoplastics resins for innumerousapplications such as rigid or flexible packaging materials, blow moldingand injection molding.

Propylene is typically obtained in large quantity scales as a byproductof catalytical or thermal oil cracking, or as a co-product of ethyleneproduction from natural gas. (Propylene, Jamie G. Lacson, CEH MarketingResearch Report-2004, Chemical Economics Handbook-SRI International).The use of alternative routes for the production of propylene has beencontinuously evaluated using a wide range of renewable raw materials(“Green Propylene”, Nexant, January 2009). These routes include, forexample, dimerization of ethylene to yield butylene, followed bymetathesis with additional ethylene to produce propylene. Another routeis biobutanol production by sugar fermentation followed by dehydrationand methatesis with ethylene. Some thermal routes are also beingevaluated such as gasification of biomass to produce a syngas followedby synthesis of methanol, which may then produce green propylene viamethanol-to-olefin technology.

Propylene production by iso-propanol dehydration has been well-describedin document EP00498573B1, wherein all examples show propyleneselectivity higher than 90% with high conversions. Dehydration of1-propanol has also been studied in the following articles: “Mechanismand Kinetics of the Acid-Catalyzed Dehydration of 1- and iso-propanol inHot Compressed Liquid Water” (Antal, M et al., Ind. Eng. Chem. Res.1998, 37, 3820-3829) and “Fischer-Tropsch Aqueous Phase Refining byCatalytic Alcohol Dehydration” (Nel, R. et al., Ind. Eng. Chem. Res.2007, 46, 3558-3565). The reported yield is higher than 90%.

EXAMPLES Example 1 Engineering of Acetoacetyl-CoA Hydrolase

An enzyme having acetoacetyl-CoA transferase activity may be engineeredby any method known in the art to produce an acetoacetyl-CoA specifichydrolase.

In an exemplary method, an amino acid sequence of an enzyme havingacetoacetyl-CoA transferase activity is obtained. Next, the glutamicacid residue at a position corresponding to amino acid position 51 ofSEQ ID NO: 1 in the enzyme is identified by aligning the amino acidsequence of the enzyme with SEQ ID NO: 1. A site in the enzymecorresponding to amino acid position 51 of SEQ ID NO: 1 is then selectedfor substitution. Such substitution of the identified glutamic acidresidue may include substitution of the glutamic acid residue foraspartic acid and may be made by any method known in the art including,for example, site directed mutagenesis. Subsequently, an acetoacetyl-CoAspecific hydrolase is obtained having a specific acetoacetyl-CoAhydrolase activity at least 10× higher than its acetyl-CoA hydrolaseactivity.

Example 2 Modification of Microorganism for Production of 1-Propanol and2-Propanol

A microorganism such as a bacterium is genetically modified to produce1-propanol and 2-propanol from a fermentable carbon source including,for example, glucose.

In an exemplary method, a microorganism may be genetically engineered byany methods known in the art to comprise: i.) one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of the fermentable carbon source todihydroxyacetone-phosphate or glyceraldehyde 3-phosphate and one or morepolynucleotides coding for enzymes in a pathway that catalyze aconversion of dihydroxyacetone-phosphate or glyceraldehyde 3-phosphateto 1-propanol and 2-propanol.

Alternatively, a microorganism that lacks one or more enzymes (e.g., oneor more functional enzymes that are catalytically active) for theconversion of a fermentable carbon source to 1-propanol and 2-propanolmay be genetically modified to comprise one or more polynucleotidescoding for enzymes (e.g., functional enzymes including, for example anyenzyme disclosed herein) in a pathway that the microorganism lacks tocatalyze a conversion of the fermentable carbon source to 1-propanol and2-propanol.

Example 3 Fermentation of Glucose by Genetically Modified Microorganismto Produce 1-Propanol and 2-Propanol

A genetically modified microorganism, as produced in Example 1 above,may be used to ferment a carbon source to produce 1-propanol and2-propanol.

In an exemplary method, a previously-sterilized culture mediumcomprising a fermentable carbon source (e.g., 9 g/L glucose, 1 g/LKH2PO4, 2 g/L (NH4)2HPO4, 5 mg/L FeSO4.7H2O, 10 mg/L MgSO4.7H2O, 2.5mg/L MnSO4.H2O, 10 mg/L CaCl2.6H2O, 10 mg/L CoCl2.6H2O, and 10 g/L yeastextract) is charged in a bioreactor.

During fermentation, anaerobic conditions are maintained by, forexample, sparging nitrogen through the culture medium. A suitabletemperature for fermentation (e.g., about 30° C.) is maintained usingany method known in the art. A near physiological pH (e.g., about 6.5)is maintained by, for example, automatic addition of sodium hydroxide.The bioreactor is agitated at, for example, about 50 rpm. Fermentationis allowed to run to completion.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent disclosure. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the disclosure and does not pose alimitation on the scope of the disclosure otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the disclosure.

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group can be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the disclosureto be practiced otherwise than specifically described herein.Accordingly, this disclosure includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by thedisclosure unless otherwise indicated herein or otherwise clearlycontradicted by context.

Specific embodiments disclosed herein can be further limited in theclaims using consisting of or and consisting essentially of language.When used in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the disclosure so claimed areinherently or expressly described and enabled herein.

It is to be understood that the embodiments of the disclosure disclosedherein are illustrative of the principles of the present disclosure.Other modifications that can be employed are within the scope of thedisclosure. Thus, by way of example, but not of limitation, alternativeconfigurations of the present disclosure can be utilized in accordancewith the teachings herein. Accordingly, the present disclosure is notlimited to that precisely as shown and described.

While the present disclosure has been described and illustrated hereinby references to various specific materials, procedures and examples, itis understood that the disclosure is not restricted to the particularcombinations of materials and procedures selected for that purpose.Numerous variations of such details can be implied as will beappreciated by those skilled in the art. It is intended that thespecification and examples be considered as exemplary, only, with thetrue scope and spirit of the disclosure being indicated by the followingclaims. All references, patents, and patent applications referred to inthis application are herein incorporated by reference in their entirety.

1) An engineered enzyme having acetoacetyl-CoA substrate specificity andacetoacetyl-CoA specific hydrolase activity. 2) The engineered enzyme ofclaim 1, wherein the engineered enzyme comprises i) an amino acidsequence of an enzyme having acetoacetyl-CoA transferase activity andii) a substitution of a glutamic acid residue to an aspartic acidresidue at a position corresponding to amino acid position 51 of SEQ IDNO:
 1. 3) The engineered enzyme of claim 2, wherein the enzyme havingacetoacetyl-CoA transferase belongs to an enzyme family having 3-oxoacidCoA-transferase activity. 4) The engineered enzyme of claim 2, whereinthe enzyme having acetoacetyl-CoA transferase activity isbutyrate-acetoacetate CoA-transferase or acetate-acetoacetate-CoAtransferase. 5) The engineered enzyme of claim 2, wherein the enzymehaving acetoacetyl-CoA transferase activity is from Clostridiumacetobutilicum or Escherichia coli. 6) The engineered enzyme of claim 1,wherein the engineered enzyme has the amino acid sequence as set forthin SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:
 6. 7) The engineered enzymeof claim 1, wherein the engineered enzyme has a specific acetoacetyl-CoAhydrolase activity at least 10× higher than its acetyl-CoA hydrolaseactivity. 8) An engineered enzyme having the amino acid sequence as setforth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:
 6. 9) A modifiedmicroorganism comprising one or more polynucleotides coding for one ormore enzymes in a pathway with acetoacetate as an intermediate orend-product, and an engineered enzyme having acetoacetyl-CoA substratespecificity and acetoacetyl-CoA specific hydrolase activity. 10) Themodified microorganism of claim 9, wherein the engineered enzymecomprises i) an amino acid sequence of an enzyme having acetoacetyl-CoAtransferase activity and ii) a substitution of a glutamic acid residueto an aspartic acid residue at a position corresponding to amino acidposition 51 of SEQ ID NO:
 1. 11) The modified microorganism of claim 10,wherein the enzyme having acetoacetyl-CoA transferase activity is froman enzyme family having 3-oxoacid CoA-transferase activity. 12) Themodified microorganism of claim 10, wherein the enzyme havingacetoacetyl-CoA transferase activity is butyrate-acetoacetateCoA-transferase or acetate-acetoacetate-CoA transferase. 13) Themodified microorganism of claim 10, wherein the enzyme havingacetoacetyl-CoA transferase activity is from Clostridium acetobutilicumor Escherichia coli. 14) The modified microorganism of claim 9, whereinthe enzyme has the amino acid sequence as set forth in SEQ ID NO: 2, SEQID NO: 4, or SEQ ID NO:
 6. 15) The modified microorganism of claim 9,wherein the engineered enzyme has a specific acetoacetyl-CoA hydrolaseactivity at least 10× higher than its acetyl-CoA hydrolase activity. 16)The modified microorganism of claim 9, wherein the microorganism has adisruption in one or more polynucleotides that code for one or moreenzymes that decarboxylate pyruvate or a disruption in one or morepolynucleotides that code for a transcription factor of an enzyme thatdecarboxylates pyruvate. 17) The modified microorganism of claim 16,wherein the disruption in the one or more enzymes that decarboxylatepyruvate is a deletion or a mutation. 18) The modified microorganism ofclaim 17, wherein the one or more enzymes that decarboxylate pyruvateinclude pdc1, pdc 5, and/or pdc6, and wherein the one or moretranscription factors of the one or more enzymes that decarboxylatepyruvate include pdc2. 19) A method of engineering an enzyme havingacetoacetyl-CoA substrate specificity and acetoacetyl-CoA specifichydrolase activity, the method comprising: a) selecting an enzyme havingacetoacetyl-CoA transferase activity, and b) substituting a glutamicacid residue to an aspartic acid residue at a position corresponding toamino acid position 51 of SEQ ID NO: 1 in the enzyme havingacetoacetyl-CoA transferase activity to produce an engineered enzyme.20) The method of claim 19, wherein the substitution is introduced viasite directed mutagenesis. 21) The method of claim 19, wherein theenzyme having acetoacetyl-CoA transferase activity is from an enzymefamily having 3-oxoacid CoA-transferase activity. 22) The method ofclaim 19, wherein the enzyme having acetoacetyl-CoA transferase activityis butyrate-acetoacetate CoA-transferase or acetate-acetoacetate-CoAtransferase. 23) The method of claim 19, wherein the enzyme havingacetoacetyl-CoA transferase activity is from Clostridium acetobutilicumor Escherichia coli. 24) The method of claim 19, wherein the engineeredenzyme has a specific acetoacetyl-CoA hydrolase activity at least 10×higher than its acetyl-CoA hydrolase activity. 25) A method of producingone or more products from a fermentable carbon source, said methodcomprising: a.) providing a fermentable carbon source; and b.)contacting the fermentable carbon source with the modified microorganismof claim 9 in a fermentation media, wherein the microorganism producesone or more products from the fermentable carbon source. 26) The methodof claim 25, wherein the carbon source is contacted with the modifiedmicroorganism under anaerobic conditions.